This research focuses on the modeling of dendrimer molecules for their application as delivery vectors within drug and gene therapy systems. We examine how the architecture and composition of dendrimers affect their drug and gene binding efficacies along with their interactions with anionic bilayers. We specifically focus on how the weakly basic nature of dendrimer monomers and the addition of neutral grafts to dendrimer surface groups affect their interactions with drugs, linear polyelectrolytes, and bilayers. By using polymer self-consistent field theory (SCFT) to model such systems, we develop a computationally efficient means to provide physical insights into these systems, which are intended to guide dendrimer design for delivery applications.We study the conformational properties of weakly basic (annealed) polyelectrolyte dendrimers by developing a SCFT model that explicitly accounts for the acid-base equilibrium reaction of the weakly basic monomers. We specifically focus on the role of local counterion concentration upon the charge and conformations of the annealed polyelectrolyte dendrimers. We compare our results to existing polymer scaling theories and develop a strong stretching theory for the dendrimer molecules.We extend the previous study to model the interactions between weakly basic dendrimers and weakly acidic, hydrophobic drug molecules. We specifically examine the effects of excluded volume, electrostatic, and enthalpic interactions on the binding efficacies between dendrimers and drugs under a variety of dendrimer generations, solution pOH conditions, drug sizes, and Bjerrum length values.We study the role of neutral dendrimer grafts on the conformations and drug binding efficacies of dendrimers. We then elucidate how the observed conformational changes affect the charge of the dendrimers. Furthermore, we examine how the presence of grafts affects the steric, electrostatic, and hydrophobic interactions between the drugs and dendrimers under a variety of solution conditions. We compare our results with the binding efficacies observed for non-grafted dendrimers to delineate the conditions under which the grafted dendrimers are better suited as drug hosts.We include semi-flexible, anionic linear polyelectrolyte (LPE) molecules in our grafted dendrimer SCFT framework to model the interactions between dendrimers and negatively charged genetic materials. Specifically, we examine how neutral dendrimer grafts, LPE stiffness, and solution pOH affect the interactions between dendrimers and LPEs. We then use our SCFT potential fields as input into Monte Carlo simulations in order to determine the dendrimer-LPE potentials of mean force and the resulting loop and tail statistics of the dendrimer-adsorbed LPE chains.We incorporate a negatively charged bilayer into our grafted dendrimer SCFT framework to model dendrimer interactions with a cellular membrane. We specifically examine the role of dendrimer grafting length, solution pH, and membrane tension on such interactions. By comparing our results with SCFT calculations of fixed dendrimer conformations and hard sphere nanoparticles in the presence of membranes, we delineate the role of dendrimer flexibility and porosity on the interactions between dendrimers and anionic bilayers. / text

published_or_final_version / Surgery / Master / Master of Philosophy

'Regulators of G protein signaling' (RGS proteins) modulate the G proteincycle by enhancing the GTPase activity of Ga subunits. These changesaccelerate the kinetics of ion channel modulation by Gai/o-coupled receptors(GPCRs) such as the G protein-gated inward rectifier K+ (GIRK/Kir3) channel. Myexperiments indicate that a single cerebellar granule (CG) neuron, a cell type thatendogenously expresses GIRK channels is able to express a wide variety ofRGS proteins. I selected two of them, which are widely expressed andtranscriptionally regulated during pathophysiologic conditions, to compare theirfunctional properties. I originally described the differential modulatory effects oftwo RGS proteins, the RGS3 short isoform (RGS3s) and RGS4, on muscarinicm2 and serotonin 1A receptor-coupled Kir3.1/Kir3.2a channels expressed inChinese hamster ovary (CHO-K1) cells. Both RGS3s and RGS4 acceleratedGIRK activation and deactivation current kinetics in a similar way. However, onlyRGS3s si gnificantly decreased the maximal GIRK current (Imax) elicited by ACh(~45% inhibition) and significantly increased the EC50 for both GPCRs. Thehypothesis that emerged from this initial study was that the distinct RGS4 Nterminaldomain mediated a direct coupling of RGS4 to GPCR-GIRK channelsignaling complexes that was not shared by RGS3s. To test this hypothesis, Iepitope-tagged several GPCRs, the Kir3.1 subunit, RGS3s, RGS4, and severaldeletion mutants and chimeras for co-immunoprecipitation experiments. Using anepitope-tagged degradation resistant RGS4 mutant RGS4(C2V), I detected coprecipitationof different GPCR-GIRK channel complexes with RGS4 but notRGS3s.The functional impact of RGS4 coupling to the GPCR-Kir3 channelcomplex versus uncoupled RGS3s was not apparent in recordings from CHO-K1cells presumably due to a high degree of RGS collision-coupling. Controlledexpression in Xenopus oocytes revealed a 30-fold greater potency for RGS4 inthe accelerating GIRK channel gating kinetics. In summary, these findings demonstrate that one of the ways for the cellto achieve signaling pathway specificity may be through selective coupling of thedifferent GPCR-effector-RGS protein complexes.

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.

A series of pyridinium cationic lipids was designed, synthesized and characterized. These lipids varied in the lipophilic part, bearing C9 to C20 saturated, unsaturated, straight and branched hydrocarbon chains. The lipid shape parameter was calculated from the molecular structure of these lipids based on the partial molar volumes of the atoms, and standard bond lengths and bond angles, using fragment additive methods. The shape parameter controls the lamellar/hexagonal phase balance in lipoplexes of the lipid with deoxyribonucleic acid (DNA). The lipid phase behaviour of the lipoplexes was derived from small-angle X-ray scattering experiments and was successfully correlated with the calculated lipid shape parameter. The synthesized pyridinium lipids were co-formulated (1:1) with 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC) as the co-cationic lipid in 1:1 ratio, and the mixed cationic lipids were co-formulated (3:2) with the neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or cholesterol. The effect of variation in cationic lipid structure and lipoplex formulation on the transfection of nucleic acid (β-galactosidase and green fluorescent protein (GFP)) into CHO-K1 cells and the cytotoxicity of these formulations was assessed. Initial studies on the synthesized lipids bearing saturated and terminally unsaturated C16 chains showed that a Transfection Index (TIPSV) which encompasses the variation in the lipid shape parameter, the phase packing in a hexagonal lipoplex and the partition of these lipids into the lipoplex successfully correlated with transfection efficiency. To further investigate the effect of the variation of the partition of these lipids to the lipoplex, transfection studies were performed on a series of pyridinium lipids with straight saturated and unsaturated chains of varied lengths, with similar shape parameters but varied partition coefficients (clogP). The correlation of these experimental transfection data with the initial TIPSV was unsuccessful, but the data suggested that chain length as it relates to chain mixing and chain melting behaviours of pure lipids played a role in transfection. A refined transfection index (TIPSVM) was proposed which contained terms for the lipid shape parameter, the phase packing into a hexagonal lipoplex, the partition of these lipids into the lipoplex and a chain melting term. TIPSVM gave an acceptable correlation with the experimental transfection efficiency for the range of compounds. Additional experimental transfection data were obtained for compounds with widely variable lipid shape parameters, either as pure compounds, blends of two pure compounds, or statistically produced mixtures of mixed-chain compounds. Although very short-chain compounds (C9) and very lipophilic compounds (C20) performed poorly, the results from the blends allow the assessment of the role of the shape parameter in the TI. Since the shape parameter and the volume filling term are both calculated with the same molecular parameter, the experimental work demonstrated that only one of these terms is required. Thus a three parameter transfection index (TIPVM) was proposed and found to correlate with the entire set of comparable data. A Quantitative structure–activity relationship (QSAR) study was done on the cytotoxicity of the transfection formulations utilized. The toxicity of the synthesized pyridinium lipids was shown to correlate with the shape parameter, the lipid mixture partition co-efficient (clogP) and the charge ratio of the lipoplex formulation. Taken together, the developed transfection index TIPVM and the cytotoxicity correlation uncovered can be used in the design of low-toxicity, high activity pyridinium lipids for transfection of DNA. / Graduate / pariapz@uvic.ca

No description available.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The transfer and expression of genes in cells is an important technique for basic research and gene therapy of human disease. A model for gene therapy has been investigated making use of a transfection complex consisting of three components, the DNA i.e. the gene to be transferred and expressed; a gene delivery vehicle viz. a cationic liposome and a cell specific targeting ligand, asialoorosomucoid (AOM). Cationic liposomes are positively charged liposomes that have been prepared from synthetic lipids and have been shown to complex or bind to DNA via electrostatic attraction. They have shown potential as an efficient non-viral gene delivery vehicle in human gene therapy. In this investigation, a novel cationic liposome consisting of 3B [N -(N',N'-dimethylaminopropane)carbamoyl] cholesterol (Chol-T), dioleoylphosphatidylethanolamine (DOPE) and biotinylcholesteryl formylhydrazide was prepared and assessed as a mediator of DNA delivery in a mammalian cell culture system viz. the HepG2 cell line. The cationic liposome was synthesised and characterised by electron microscopy. Foreign DNA may be specifically delivered to target cells by a carrier system which makes use of the recognition of the asialoglycoprotein AOM by cognate receptors on the HepG2 cell plasma membrane. The positively charged AOM was biotinylated and due to this biotinylation, binds streptavidin which contains specific binding sites for biotin. The cationic liposome itself contains biotin residues in its bi-Iayer which in turn binds streptavidin resulting in a ternary complex. Further, due to the DNA binding capability of the cationic liposome, a transfection complex is produced consisting of the three components. The experiments were based on the following concepts: (i) Hepatocytes possess a unique receptor that binds to and internalises galactose-terminal asialoglycoproteins by receptor mediated endocytosis. (ii) Due to electrostatic attraction, DNA binds to cationic liposomes forming soluble complexes. (iii) Through the biotin-streptavidin reaction, the biotinylated AOM is attached to the cationic liposome containing biotin forming complexes enabling targeted delivery of the DNA. (iv) DNA containing the pGL3 gene for the luciferase enzyme was used and following transfection experiments, the luciferase assay was performed to ensure successful transfection. The complexes were tested on the hepatocellular carcinoma cell line, HepG2, which possess the asialoglycoprotein receptor. Transfection studies were conducted using a transient expression system, the luciferase assay system. Some degree of success in the transfection of HepG2 cells was observed. Results obtained in this study suggest that transfection using our targeted transfection complex consisting of cationic liposomes and cell specific targeting ligands does in fact transfect cells by receptor mediation. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, 2006.

Enteroviruses belong to the Picornaviridae family and are small icosahedral viruses with RNA genomes of positive polarity, containing a single open reading frame. They mostly cause mild or asymptomatic infections, but also a wide array of diseases including: poliomyelitis, encephalitis, gastroenteritis, aseptic meningitis, myocarditis, hand-foot-and-mouth disease, hepatitis and respiratory diseases, ranging from severe infections to the common cold. The projects described in this thesis have been carried out through reverse genetic studies of Enterovirus B and Rhinovirus C.                   In Papers I and II, a cassette vector was used to study recombination and translation of the RNA genome. It was found that the non-structural coding region could replicate when combined with the structural protein-coding region of other viruses of the same species. Furthermore, the genome could be translated and replicated without the presence of the structural protein-coding region. Moreover, it was found that when two additional nucleotides were introduced, shifting the reading frame, the virus could revert to the original reading frame, restoring efficient replication. In Paper III, a vector containing the genome of echovirus 5 was altered to produce an authentic 5’end of the in vitro transcribed RNA, which increased efficiency of replication initiation 20 times. This result is important, as it may lead to more efficient oncolytic virotherapy. An authentic 5’end was further used in Paper IV, where replication of Rhinovirus C in cell lines was attempted. Although passaging of the virus was unsuccessful, the genome was replicated and cytopathic effect induced after transfection. The restriction of efficient replication was therefore hypothesized to lie in the attachment and entry stages of the replication cycle. In Paper V, a cytolytic virus was found to have almost 10 times larger impact on gene expression of the host cell than a non-cytolytic variant. Furthermore, the lytic virus was found to build up inside the host cell, while the non-cytolytic virus was efficiently released.                   As a whole, this thesis has contributed to a deeper understanding of replication of enteroviruses, which may prove important in development of novel vaccines, antiviral agents and oncolytic virotherapies.

No description available.